Developing screens to identify new biologically active compounds can present unique and difficult challenges, especially when screening naturally occurring complex biological materials (sometimes referred to as "natural samples" or "natural products"), various biological preparations, chemical mixtures, and other complex materials. Major problems include low concentrations of active compounds, unknown components that can interfere with screening agents, and isolation of the new compound once a positive sample is obtained. Despite these obstacles, the pharmaceutical industry still maintains a strong interest in the screening of complex mixtures. For example, it is widely recognized that nature provides a virtually endless supply of new chemical structures that are often difficult or impossible to synthesize in a cost-effective manner. Most natural products have some bio-activity, and historically, natural products and their analogs have been the most successful source of therapeutic compounds.
Screening technologies for therapeutic and other biologically active compounds fall into two broad categories: bioassays and mechanism-based assays (Gordon et al., J. Med. Chem. 37:1386-1401, 1994).
Bioassays represent the oldest, and so far, most productive screening tool. Bioassays measure the effect of natural samples on the viability or metabolism of disease-related cell types such as bacteria, fungi, viruses, and tumor cells. For example, the .beta.-lactam antibiotics (e.g., penicillins and cephalosporins) were discovered by testing microbial broths for bacterial growth inhibition in culture tests. Likewise, the antifungal compounds, nystatin and amphotericin B, were isolated from broths that inhibited yeast growth in culture tests. However, mainly due to the lack of specificity and sensitivity of bioassays, the more sophisticated mechanism-based assays have replaced most bioassays as primary screens.
Mechanism-based assays can be subdivided into three general categories: (1) recombinant cell-based assays, (2) enzymatic/biochemical assays, and (3) binding assays. Today's assays must satisfy the need for high throughput capacity, so they must be robust, simple, and amenable to automation in a parallel processing mode.
Recombinant, cell-based assays screen for a given, known functional response. Usually a target receptor, enzyme, or other protein is introduced into cultured cells by genetic engineering. Inhibition or induction of target activity is associated with an easily-measured response. For example, modifiers of transcription factors (TF) can be measured by fusing the TF's target DNA sequence (typically an enhancer or promoter region) to a luciferase (light-producing) gene. TF agonists result in transcription of the luciferase gene, and light is produced. If an antagonist is present, light is not produced. One advantage of cell-based assays over enzymatic and binding assays is that they may provide more physiologically appropriate leads, because intact cells are used. On the other hand, cell-based screens can be very difficult to develop, slow and quite variable in their results (Janzen et al., Society for Biomolecular Screening Meeting, Nov. 7-10, 1995).
Enzymatic assays are cell-free screens that directly or indirectly test the effect of soluble compounds on the activity of purified target enzymes that are related to disease processes. For example, viral reverse transcriptase inhibitors can be screened by measuring the incorporation of radiolabeled thymidine into a growing DNA chain from a polyuridine RNA template. These assays can be very sensitive and are amenable to automation using microtiter plates. For natural product screening, however, unknown compounds in the samples can dramatically interfere with screening results, leading to unacceptably high levels of false negatives and false positives. For example, greater than 15% of aqueous extracts from terrestrial plants, cyanobacteria, marine invertebrates, and algae exhibit positive activity in screens for anti-HIV compounds, due to interfering compounds such as plant tannins (Cardellina et al., J. Nat. Prod. 56:1123-1129, 1993).
Binding assays are particularly useful for screening soluble mixtures of biological or chemical materials for compounds that bind, and thus potentially modulate or inhibit, physiologically active target molecules. These assays have been major screening tools in the drug discovery efforts of pharmaceutical and biotechnology companies. In immobilized-target binding assays, the target molecule (usually a protein) can be affixed or tethered to a solid substrate such as the side of a microtiter well, beads, or chromatographic supports. If the target molecule is a receptor, it can be expressed on the membrane of a cell attached to the solid support. The samples are incubated with the immobilized targets, and bound ligands are detected, usually through an associated calorimetric or fluorescent reaction. Alternatively, the sample is mixed with a soluble-phase target that is captured using an anti-target antibody. Such binding assays are advantageous because they facilitate the washing and isolation of target-ligand complexes.
However, immobilized-target binding assays also suffer from several disadvantages, particularly as a method for screening natural biological samples for new active compounds. One problem is that the binding of multiple background compounds, if present in sufficient quantities, may produce a positive signal that is indistinguishable from that of a single potential therapeutic compound. Therefore, screening with immobilized-target binding assays often requires heavy washing or improved clean-up capability. Another general problem is that affixing target proteins to solid substrates often inactivates the protein or produces a functional change. This problem can be addressed to some extent by using recombinant DNA technology to insert an inert "handle" such as a peptide epitope into the target protein. The protein-ligand complex can then be isolated through the use of an antibody to this epitope. However, development of these modified targets is time-consuming and expensive.
One commonly used binding assay is the microtiter-format, enzyme-linked immunosorbent assay (ELISA). One disadvantage is that the target molecule, which is usually attached to the well wall, does not contact most of the soluble sample dispersed throughout the well. Therefore, greater reaction times are needed, although some improvements have been made through using reduced reaction volumes. Another problem is that an ELISA requires the development of specific monoclonal antibodies, a time-consuming and often unsuccessful process.
There remains a need for rapid and cost-effective screening tools for discovering new bioactive compounds and potential drugs that bond to essential molecules of key metabolic pathways. The present invention relates to an improved method of screening complex biological material for new active compounds using capillary electrophoresis.